Methods of determining the amount of microorganisms present in a test sample

ABSTRACT

Methods of determining the amount of microorganisms present in a test sample. The methods include a) incubating the test sample with a growth media to form an incubated sample, wherein the growth media includes an enzyme substrate and the enzyme substrate includes an enzymatically hydrolyzable group and a fluorescent group, wherein microorganisms present in the test sample include an enzyme that hydrolyzes the hydrolyzable group from the fluorescent group to form a fluorescently detectable product, wherein the fluorescently detectable product has both an acidic and basic species; b) exciting the fluorescently detectable product with light having a wavelength of Exλiso for a time sufficient for the fluorescently detectable product to emit light, wherein Exλiso is the absorbance isosbestic point of the fluorescently detectable product; c) detecting light emitted at a wavelength of Emλ1; and d) quantifying the light emitted at the wavelength of Emλ1, wherein the quantity of the light emitted at the wavelength Emλ1 is indicative of the amount of microorganisms present in the test sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofPCT/US2010/060936, filed Dec. 17, 2010, which claims priority to U.S.Provisional Application No. 61/288,883, filed Dec. 22, 2009, thedisclosure of which is incorporated by reference in its/their entiretyherein.

TECHNICAL FIELD

The present disclosure relates to methods of detecting microorganismsutilizing fluorogenic compounds.

BACKGROUND

7-hydroxycoumarin-based dyes (also called umbelliferones) and theirderivatives are widely used as indicators for enzyme activity. Oneexample of an umbelliferone is 4-methylumbelliferone (referred to as4-MU), which is used for the detection of coliforms, amongst otherthings. The highest fluorescence of 4-MU (and other umbelliferones) isobserved from the basic form, therefore such detection has to beundertaken at a pH of 8 to 10.

SUMMARY

There are numerous situations in which a detection response is desiredat lower pH values. For example, kinetic studies of acid phosphatasesrequire real-time detection of enzyme activity in acidic media. Theability to observe fluorescence at acidic pHs would also eliminatenecessary steps in protocols, thereby making analyses quicker andeasier. Because of the desire to carry out low pH fluorescence analysis,there remains a need for analysis methods that can be carried outirrespective of pH.

Disclosed herein is a method of detecting microorganisms in a testsample, the method including the steps of: a) incubating the test samplewith an enzyme substrate to form an incubated sample, wherein the enzymesubstrate includes an enzymatically hydrolysable group and a fluorescentgroup, wherein microorganisms present in the test sample include anenzyme that hydrolyzes the hydrolysable group from the fluorescent groupto form a fluorescently detectable product, wherein the fluorescentlydetectable product has both an acidic and basic species; b) exciting thefluorescently detectable product with light having a wavelength ofExλiso for a time sufficient for the fluorescently detectable product toemit light, wherein Exλiso is the absorbance isosbestic point of thefluorescently detectable product; and c) detecting light emitted at awavelength of Emλ1.

Also disclosed is a method of detecting microorganisms in a test sample,the method including the steps of: a) incubating the test sample with anenzyme substrate, wherein the enzyme substrate includes an enzymaticallyhydrolysable group and a fluorescent group, wherein microorganismspresent in the test sample include an enzyme that hydrolyzes thehydrolysable group from the fluorescent group to form a fluorescentlydetectable product, wherein the fluorescently detectable product hasboth an acidic and basic species; b) exciting the fluorescentlydetectable product with light having a wavelength of Exλiso for a timesufficient for the fluorescently detectable product to emit light,wherein Exλiso is the isosbestic point of the fluorescently detectableproduct; c) detecting light emitted at a wavelength of Emλ1 as a resultof the excitation with light having a wavelength of Exλiso; d) excitingthe fluorescently detectable product with light having a wavelength ofExλ2 for a time sufficient for the fluorescently detectable product toemit light, wherein Exλ2 is the absorption maximum of the basic speciesof the fluorescently detectable product or the absorption maximum of theacidic species of the fluorescently detectable product; e) detectinglight emitted at a wavelength of Emλ1 as a result of the excitation withlight having a wavelength of Exλ2; and f) calculating a ratio based onthe light emitted as a result of the Exλiso excitation light and thelight emitted as a result of the Exλ2 excitation light, wherein theratio is indicative of the amount of microorganisms present in the testsample.

Also disclosed is a kit for testing for the presence of microorganismsin a test sample, the kit including: enzyme substrate that includes anenzymatically hydrolysable group and a fluorescent group, whereinmicroorganisms present in the test sample include an enzyme thathydrolyzes the hydrolysable group from the fluorescent group to form afluorescently detectable product wherein the fluorescently detectableproduct has both an acidic and basic species; and a light source capableof providing light having a wavelength of Exλiso, wherein Exλiso is theisosbestic point of the fluorescently detectable product, and whereinexcitation of the fluorescently detectable product with light having awavelength of Exλiso causes the fluorescently detectable product to emitlight.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure may be more completely understood in consideration of thefollowing detailed description of various embodiments of the disclosurein connection with the accompanying drawings, in which:

The figures are not necessarily to scale. Like numbers used in thefigures refer to like components. However, it will be understood thatthe use of a number to refer to a component in a given figure is notintended to limit the component in another figure labeled with the samenumber.

FIGS. 1A, 1B, and 1C are the absorption spectra of 4-methylumbelliferone(4-MU) for different pH values (FIG. 1A), the emission (excitation 455nm) as a function of pH for two different excitation wavelengths (FIG.1B), and emission spectra from 400 nm to 500 for different pH values(FIG. 1C);

FIG. 2 is a flowchart depicting exemplary methods disclosed herein;

FIG. 3 is a flowchart depicting exemplary methods disclosed herein;

FIG. 4 is a flowchart depicting exemplary methods disclosed herein;

FIGS. 5A, 5B, and 5C are an emission spectra (375 nm) as a function ofpH (FIG. 5A), an emission spectra (455 nm) as a function of pH at threedifferent excitation wavelengths (FIG. 5B), and a plot of the ratios ofpeak emissions for excitation at different wavelengths (FIG. 5C);

FIG. 6 is an emission (450 nm) spectra for7-hydroxycoumarin-3-carboxylic acid ethyl ester (EHC) as a function ofpH;

FIGS. 7A, 7B, and 7C are an absorption spectra of3-(2-thineyl)umbelliferone (TU) for different pH values (FIG. 7A), 380nm (FIG. 7B), and emission (at 500 nm) upon excitation at 405 nm andemission (at 490 nm) upon excitation at 380 nm as a function of pH (FIG.7C);

FIGS. 8A, and 8B are emission (at 455 nm) upon excitation at 335 nm as afunction of pH for 4-MU and 4-MUG in the presence and absence ofβ-D-galactosidase (FIG. 8A), and emission (at 455 nm) upon excitation at360 nm as a function of pH for 4-MU and 4-MUG in the presence andabsence of β-D-galactosidase (FIG. 8B);

FIG. 9 is emission at 445 nm (excitation 370 nm or 400 nm) as a functionof pH for MHCgal in the presence and absence of β-D-galactosidase; and

FIG. 10 is emission at 490 nm upon excitation at 380 nm as a function ofpH for 3-(2-thienyl)umbelliferone-β-D-galactopyranoside (TUgal) in thepresence and absence of β-D-galactosidase, and emission at 500 nm uponexcitation at 410 nm as a function of pH for TUgal in the presence andabsence of β-D-galactosidase.

DETAILED DESCRIPTION

In the following description, reference is made to the accompanyingdrawing that forms a part hereof, and in which are shown by way ofillustration several specific embodiments. It is to be understood thatother embodiments are contemplated and may be made without departingfrom the scope or spirit of the present disclosure. The followingdetailed description, therefore, is not to be taken in a limiting sense.

All scientific and technical terms used herein have meanings commonlyused in the art unless otherwise specified. The definitions providedherein are to facilitate understanding of certain terms used frequentlyherein and are not meant to limit the scope of the present disclosure.

Unless otherwise indicated, all numbers expressing feature sizes,amounts, and physical properties used in the specification and claimsare to be understood as being modified in all instances by the term“about.” Accordingly, unless indicated to the contrary, the numericalparameters set forth in the foregoing specification and attached claimsare approximations that can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings disclosed herein.

The recitation of numerical ranges by endpoints includes all numberssubsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3,3.80, 4, and 5) and any range within that range.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” encompass embodiments having pluralreferents, unless the content clearly dictates otherwise. As used inthis specification and the appended claims, the term “or” is generallyemployed in its sense including “and/or” unless the content clearlydictates otherwise.

Disclosed herein are methods and kits for detecting microorganisms insamples. In embodiments, a method may generally include the steps ofincubating the test sample with the enzyme substrate, irradiating theincubated sample with a first wavelength, and detecting light emittedfrom the sample. An enzyme substrate is a compound that can be cleavedby an enzyme of a microorganism, forming a fluorescently detectableproduct. The fluorescently detectable product is then irradiated at awavelength of the isosbestic point of the fluorescently detectableproduct. Upon excitation of the fluorescently detectable product, thefluorescently detectable product will emit light, which can then bedetected to confirm the presence of the microorganism in the testsample.

In embodiments, microorganisms that can be detected using methods andkits as disclosed herein can include bacteria, and fungi for example.Exemplary fungi include both yeasts (including for example,Saccharomyces cerevisiae, Cryptococcus neoformans, Candida albicans,Candida tropicalis, Candida stellatoidea, Candida glabrata, Candidakrusei, Candida parapsilosis, Candida guilliermondii, Candidaviswanathii, Candida lusitaniae, and Rhodotorula mucilaginosa) and molds(including for example, Acremonium, Aspergillus, Cladosporium, Fusarium,Mucor, Penicillium, Rhizopus, Stachybotrys, and Trichoderma) forexample. Exemplary bacteria include the following microorganisms:Aeromonas hydrophilia, Aeromonas caviae, Aeromonas sobria, Bacilluscereus, Bacillus stearothermophilus, Bacillus subtilis, Bacillussphaericus, Bacteroides fragilis, Bacteroides intermedium, Citrobacterfreundii, Clostridium perfringens, Enterobacter aerogenes, Enterobactercloacae, Enterococcus faecium, Enterococcus faecalis, Escherichia coli,Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiellapneumoniae, Lactococcus lactis, Listeria monocytogenes, Listeriainnocua, Mycobacterium fortuitum, Neisseria gonorrhoeae, Organellamorganii, Peptostreptococcus anaerobius, Peptococcus magnus, Proteusmirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonaspudita, Salmonella typhimurium, Serratia liquefaciens, Serratiamarcescens, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus hominis, Staphylococcus simulans, Streptococcusagalactiae B, Streptococcus anginosus, Streptococcus constellatus,Streptococcus faecalis D, Streptococcus mutans, Streptococcus pyogenes,Streptococcus uberis, and Xanthomonas maltophilia.

Types of samples that can be tested using methods and kits as disclosedherein (which can be referred to as “test samples”) are generally notlimited. Exemplary types of samples include clinical samples,environmental samples, food samples, cosmetics, beverage samples, watersamples and soil samples. Alternatively, samples can be prepared fromarticles by rinsing the article to form a water sample to be tested, forexample. Samples such as food samples and soil samples can be digestedor subjected to other processing before disclosed methods and kits areutilized to detect microorganisms. Filtering treatments, extractiontreatments and the like can also be carried out. In embodiments where atest sample is incubated with enzyme substrate, but not growth media,pre-processing of a sample could include incubating the sample withgrowth media and then adding the resultant solution (as the test sample)to the enzyme substrate.

The test sample can be incubated with an enzyme substrate. The enzymesubstrate can be provided in or with growth media for example. Growthmedia generally are liquids or gels that are designed to support thegrowth of microorganisms. Exemplary growth media include nutrientbroths, and agar plates. The growth media can be supplied as a liquid, acondensed or dehydrated liquid, or a gel for example. An example of aproduct that supplies growth media for use via hydration is 3M™Petrifilm™ plates (3M Co., St. Paul, Minn.).

Incubating the test sample with the enzyme substrate (or enzymesubstrate and growth media) can be accomplished simply by mixing thetwo; by mixing the two and allowing the mixture to sit; by mixing thetwo and agitating the mixture; by mixing the two and heating themixture; or by mixing the two, agitating the mixture and heating themixture. The step of incubating can be undertaken for any amount oftime. In embodiments, the test sample and enzyme substrate are allowedto incubate at elevated temperatures until at least one cell divisionprocess has taken place.

In embodiments, the test sample and enzyme substrate (or enzymesubstrate and growth media) can be mixed and then agitated using anycommonly utilized mechanical agitation methods. The test sample andenzyme substrate can be mixed and then heated using any commonlyutilized heating methods. In embodiments, the test sample and growthmedia can be mixed and agitated using commonly utilized methods. Forexample, microorganisms may be detected using 3M™ Petrifilm™ plates. ThePetrifilm™ plate, which contains dehydrated growth media and enzymesubstrate, is rehydrated with the test sample, which contains themicroorganisms of interest. After addition of the test sample, therehydrated Petrifilm™ plate can be incubated at suitable conditions toenable detection of microorganisms (for example, for an aerobic count,the 3M™ Petrifilm™ plates can be placed in an incubator at 37° C. for24-48 hours; or for yeast/mold, the 3M™ Petrifilm™ plates can be placedin an incubator at 25 to 28° C. for 3 to 5 days).

An enzyme substrate as used herein is a material that is selectivelyhydrolysable by an enzyme to generate a fluorescently detectable productwhen cleaved. An enzyme substrate generally includes two portions, anenzymatically hydrolysable group (which can, but need not be abiological molecule) and a fluorescent group. An enzyme substrate can bepictorially represented by formula I below:Hydrolysable Group ------------- Fluorescent Group  (Formula I)In formula I, the moiety represented by “-------------” can be referredto as an enzymatically hydrolysable linkage. An enzymaticallyhydrolysable linkage refers to a linkage or bond that can easily becleaved by an enzyme; particularly an enzyme produced by a microorganismof interest.

The fluorescent group can be fluorescently detectable once it is cleavedfrom the enzyme substrate. It should also be noted that the enzymesubstrate can be fluorescently detectable before the fluorescent groupis cleaved from it (or stated another way, the fluorescent group can befluorescently detectable either within the enzyme substrate or cleavedfrom the enzyme substrate). In embodiments, the enzyme substrate caninclude a fluorescent dye joined to a moiety, which is cleavable by anenzyme produced by the microorganism of interest.

The hydrolysable group included in an enzyme substrate can include aglycone, a glycosyl phosphate, an ester, an amino acid or peptide, aphosphate, or a sulfate for example.

Exemplary glycone or glycosyl phosphates include α- andβ-D-galactopyranosyl, α- and β-D-glucopyranosyl, N-acetyl-α- andβ-D-galactosaminyl; N-acetyl-α- and β-glucosaminyl; β-D-glucuronyl,α-L-arabinopyranosyl, α-L-arabinofuranosyl, β-D-fucopyranosyl, α- andβ-L-fucopyranosyl, α-D-mannopyranosyl, β-D-xylopyranosyl, α-D-maltosyl,β-D-lactopyranosyl, β-D-cellobiosyl, α-D-N-acetylneuraminyl, andmyoinositol-1-yl phosphate. In embodiments, the hydrolysable group caninclude α- and β-D-galactopyranosyl, α- and β-D-glucopyranosyl, orβ-D-glucuronyl. In embodiments, the hydrolysable group includesβ-D-galactosyl or β-D-glycosyl.

Exemplary esters include butyrate, valerate, hexanoate, caprylate,octanoate, nonanoate, and palmitate. An exemplary ester can include along chain ester of umbelliferone, such as4-methylumbelliferylpalmitate, 4-methylumbelliferyllaurate,4-methylumbelliferylcaprylate, which are commercially available (suchhydrolysable groups can be used to detect esterase or palmitase).Furthermore, enzyme substrates that are hydrolysable by lipase,esterase, acylase and epoxide hydrolase can also be utilized, includingthose referred to in Sicart et al., Biotechnology Journal, 2007 2(2),221-231.

Exemplary amino acids can include carboxy terminal-linked amino acids oran acid addition salt thereof. Such amino acids can includeN-acetyl-L-lysine, L-alanine, L-arginine, L-aspartic acid,N-alpha-benzyloxycarbonyl-L-arginine, L-citrulline, gamma-L-glutamicacid, L-glycine, L-histidine, L-hydroxproline, L-isoleucine, L-leucine,L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline,L-pyroglutamic acid, L-serine, L-tryptophan, L-tyrosine, and L-valine.Exemplary peptides can include carboxy terminal-linked peptides having 1to 4 amino acids or an addition salt thereof. Such peptides can includeL-arginyl-L-arginine, N-benzyloxycarbonyl-glycyl-L-proline,L-glutaryl-glycyl-arginine, glycyl-glycine, glycyl-L-phenylalanine,glycyl-L-proline, and L-seryl-L-tyrosine. In embodiments, thehydrolysable group is a terminal-linked peptide having 1 to 4 aminoacids, wherein free amino groups optionally have a protective group, oran acid addition salt thereof.

Any fluorescent dye possessing a pH-dependent absorption and anisosbestic point (or fluorogenic portion thereof) can be utilized inenzyme substrates utilized herein. Exemplary fluorescent dyes includexanthene derivatives (such as fluorescein, rhodamine and derivativesthereof), cyanine derivatives (such as cyanine, indocarbocyanine,oxacarbocyanine, thiacarbocyanine and merocyanine and derivativesthereof), naphtalene derivatives, coumarin derivatives, oxadiazolederivatives, pyrene derivatives, oxazine derivatives, acridinederivatives, arylmethine derivatives, and tetrapyroole derivatives.

In embodiments, coumarin derivatives can be utilized in enzymesubstrates. Exemplary coumarin derivatives include 7-hydroxycoumarin(umbelliferone) derivatives. Specific 7-hydroxycoumarin derivativesinclude 4-methyl-7-hydroxycoumarin (4-methylumbelliferone or 4-MU),3-cyano-7-hydroxycoumarin (3-cyanoumbelliferone or CyU), and7-hydroxycoumarin-3-carboxylic acid esters such asethyl-7-hydroxycoumarin-3-carboxylate (EHC),methyl-7-hydroxycoumarin-3-carboxylate (MHC),3-cyano-4-methylumbelliferone, 3-(4-imidazolyl)umbelliferone, and6,8-difluoro-4-methylumbelliferone. 7-hydroxycoumarin derivatives suchas those containing a 5-membered heterocyclic ring at the 3-position canalso be utilized in fluorogenic compounds herein. Such 7-hydroxycoumarinderivatives are exemplified in U.S. Pat. No. 6,566,508 (Bentsen et al.),the disclosure of which is incorporated herein by reference thereto. Aspecific example of such a derivative is 3-(2-thienyl)umbelliferone(TU).

Exemplary enzyme substrates include, but are not limited to4-methylumbelliferone-β-D-galactopyranoside (MUG),3-cyanoumbelliferone-β-D-galactopyranoside,7-hydroxycoumarin-3-carboxylic acid ethyl ester-β-D-galactopyranoside,7-hydroxycoumarin-3-carboxylic acid methyl ester-β-D-galactopyranoside,3-(2-thienyl)umbelliferone-β-D-galactopyranoside, 4-methylumbelliferylphosphate, 3-cyanoumbelliferyl phosphate, ethyl umbelliferone-3-carboxylphosphate, methyl umbelliferone-3-carboxyl phosphate,3-(2-thienyl)umbelliferyl phosphate,5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal),5-Bromo-4-chloro-3-indolyl phosphate (BCIP), ELF 97 Acetate (Invitrogen,Carlsbad, Calif.), ELF 97 Beta D Glucuronide (Invitrogen, Carlsbad,Calif.), and ELF 97 Beta D Galactopyranoside (Invitrogen, Carlsbad,Calif.).

The fluorescently detectable product has both an acidic and a basicspecies. Generally, the acidic species of a fluorescently detectableproduct is a compound that can donate a hydrogen ion (H⁺) to anothercomponent in solution; and a basic species of a fluorescently detectableproduct is a compound that can accept a hydrogen ion (H⁺) from anothercomponent in solution. In embodiments, a fluorescently detectableproduct has an acidic species that is neutral and a basic species thatis anionic. In embodiments, a fluorescently detectable product has anacidic species that is cationic and a basic species that is neutral.

The acidic and basic species of a fluorescently detectable product canabsorb light differently (either in the amount of energy absorbed, thewavelength, or both), can emit light differently (either in intensity,the wavelength of emission, or both) or can both absorb and emit lightdifferently. FIG. 1A shows the absorbance spectra of solutions of4-methylumbelliferone (4-MU) at pHs from 2.5 to 8.0. As seen there, theabsorbance varies greatly across the monitored wavelength range (260nanometers to 440 nanometers). At a wavelength of about 332 nanometers(nm), the absorbance does not significantly vary across the entire pHrange. This wavelength, 332 nm for 4-MU, is referred to herein as theexcitation isosbestic point. The excitation isosbestic point is thewavelength at which the absorbance of the acidic and basic species ofthe fluorescently detectable product is substantially the same. Theexcitation isosbestic point of a fluorescently detectable product isalso referred to herein as Exλiso.

Excitation of a solution containing a fluorescently detectable productat the excitation isosbestic point (Exλiso) can provide a pH independentfluorescent response. In embodiments, an incubated sample can beirradiated with light having a wavelength of Exλiso. In embodiments,light having a wavelength of Exλiso can refer to light that has a peakwavelength of ±10 nm of Exλiso. In embodiments, light having awavelength of Exλiso can refer to light that has a peak wavelength of ±5nm of Exλiso. In embodiments, light having a wavelength of Exλiso canrefer to light that has a peak wavelength of ±2 nm of Exλiso.

The incubated sample can be irradiated with light having a wavelength ofExλiso for an amount of time that is sufficient for the fluorescentlydetectable product to emit light. In embodiments, the incubated samplecan be irradiated with light having a wavelength of Exλiso for fractionsof a second to tens of seconds. In embodiments, the incubated sample canbe irradiated with light having a wavelength of Exλiso for about asecond to tens of seconds. In embodiments that can be utilized with the3M™ Attest™ Biological Monitoring System (3M Co., St. Paul, Minn.), asample can be irradiated for about a second, which can be sufficienttime to obtain a stably excited sample and integrate the intensity ofemissions. In embodiments that can be utilized with the 3M™ Petrifilm™plate system (3M Co., St. Paul, Minn.), a sample can be irradiated fortens of seconds, which can be sufficient time to integrate the emissionintensity to obtain a detectable signal above background noise.

After the incubated sample is irradiated with light having a wavelengthof Exλiso for a sufficient time, the fluorescently detectable productwill emit light. Irradiation of a fluorescently detectable product witha wavelength of light that it absorbs can be referred to as excitation.Excitation at a wavelength other than Exλiso causes the acidic and basicspecies to absorb differently, and therefore the light emitted will alsobe different. This behavior yields a pH sensitive fluorescent responsefrom most fluorescently detectable products. An example of thisphenomenon can be seen in FIG. 1B. The line designated 140 shows thefluorescence (at 455 nm) of 4-methylumbelliferone (4-MU) with excitationat 360 nm at various pHs from 2.5 to 8.0. As seen there, thefluorescence changes dramatically across this pH range. Beginning at apH of 6, the fluorescence begins to increase substantially. For thisreason, it is generally accepted that fluorescence measurements of 4-MUshould be done at a basic pH (usually from a pH of 8 to 10) with anexcitation wavelength of about 360 nm so that a maximum fluorescencesignal is obtained.

The line designated 130 in FIG. 1B shows the fluorescence (at 455 nm) of4-MU with excitation at 330 nm at various pHs from 2.5 to 8.0. As seenthere, the fluorescence is almost constant across this pH range. Thecombination of the results of FIG. 1A, showing that the excitationisosbestic point (Exλiso) of 4-MU is about 332 nm; and the results ofFIG. 1B, showing that the fluorescence (at 455 nm) is substantiallyconstant across the pH range of 2.5 to 8.0 shows that excitation of asample at the excitation isosbestic point (Exλiso) can provide a pHindependent fluorescence response.

FIG. 1C shows an emission sweep (from 400 nm to 500 nm) of solutions of4-MU at pHs from 2.5 to 8.0 after excitation at 330 nm. As shown in FIG.1C, the fluorescent response is also independent of pH in that thewavelength of emission also does not vary with pH. This phenomenon canbe due to photodissociation of the neutral species (in the case of 4-MU,the acidic species) followed by proton transfer to the solvent resultingin a photogenerated anion (in the case of 4-MU, the basic species) whichthen emits at its characteristic wavelength.

Some exemplary fluorescently detectable products and their excitationisosbestic points are as follows: 4-methylumbelliferone (4-MU) has anexcitation isosbestic point (Exλiso) of about 330 nm;3-cyano-7-hydroxycoumarin (CyU) has an excitation isosbestic point(Exλiso) of about 375 nm; 7-hydroxycoumarin-3-carboxylic acid ethylester (EHC) has an excitation isosbestic point (Exλiso) of about 370 nm;7-hydroxycoumarin-3-carboxylic acid methyl ester (MHC) has an excitationisosbestic point (Exλiso) of about 370 nm; and3-(2-thienyl)umbelliferone (TU) has an excitation isosbestic point(Exλiso) of about 380 nm.

Using 4-MU as an example, shifting the excitation frequency from thecommonly utilized 360 nm to about 330 nm (Exλiso) can eliminate the 20to 30 fold decrease in fluorescence intensity when going from pH 8 to pH5. This intensity decrease can be eliminated in disclosed methodswithout requiring further steps (e.g. pH adjustment).

A pH independent fluorescence response can provide advantages in variousanalysis methods. For example, the step of adjusting the pH of the testsample to be analyzed (which step is necessary in numerous previouslyutilized methods) to a pH of 8 to 10 can be eliminated. This can renderthe analysis easier, more efficient, and more cost effective. Forexample, various types of bacteria, including for example E. Coli,Lactobacillus, and acetobacter, generate acids as they grow, a pHindependent response will ensure that results are not influenced by suchacids. For example, samples having varying acidity levels can be testedwithout the need to “neutralize” them prior to testing. For example, arelatively large difference between the excitation and emissionwavelengths can allow the use of inexpensive optical filters (forexample absorption filters as opposed to more expensive narrow bandinterference filters). For example, higher signal to noise ratios can beprovided because of greater Stokes shifts and associated tails crossing.

Excitation of the incubated sample can be accomplished using any lightsource capable of providing light having the appropriate wavelength(e.g. Exλiso). Exemplary light sources can include a visible laserdiode, a visible light emitting diode (LED), an incandescent filament,or any other suitable light source. The light source can also becombined with various filters and other optics as are commonly utilized.

In embodiments, a light source can be chosen so that a relativelyinsubstantial amount of light is present at the emission detectionwavelength. This can be advantageous because it can minimize the amountof detected light that is due to the excitation source. Alternatively,optics can be used to filter out a portion of the light from theexcitation light source. In embodiments, an excitation light source,optics, the wavelength of detection, a detector, or some combinationthereof are chosen so that substantially no light from the excitationlight source is detected and/or attributed to being an emission from afluorescently detectable product.

Once the fluorescently detectable product has been excited with lighthaving a wavelength of Exλiso for a time sufficient for thefluorescently detectable product to emit light, the emitted light isthen detected. In embodiments, the emitted light can be detected withinat least about a couple of minutes of exciting the fluorescentlydetectable product. In embodiments, the emitted light can be detectedwithin at least about a minute of exciting the fluorescently detectableproduct. In embodiments that utilize silicon based sensors (a commonchoice for NIR ranges up to 1000 nm), integration times can be about aminute if a single stage thermal electric cooling (TEC) is used to coolthe sensor to about 30° below ambient temperatures.

Detection of the light emitted by the excited fluorescently detectableproduct can be accomplished as is generally known. Detectors, such asphotomultiplier tubes, avalanche photodiodes, charge coupled devices(CCDs), photodiodes, or other active devices for example, may beutilized. The detector can also be combined with various filters andother optics as are commonly utilized.

The wavelength of emitted light to be detected can depend at least inpart on the particular fluorescently detectable product. As seen in FIG.1C, a fluorescently detectable product (such as 4-MU for example) emitslight, in various quantities, over a wide range of wavelengths. Inembodiments, the emission can be detected at a wavelength that is closeto the maximum emission. Alternatively, other wavelengths (in additionto or in place of the wavelength of maximum emission) can be monitoredin order to detect the emitted light. Emλ1 can be any desiredwavelength, and can be chosen based in part on the intensity of theemissions at various wavelengths, the particular detector (cost, etc.)that can be utilized, interference from other components in the sample(for example the enzyme substrate), or a combination thereof. Inembodiments, Emλ1 can be the wavelength at which the fluorescentlydetectable product has its maximum intensity emissions. Such an Emλ1 canbe useful to minimize possible interference from background signals,decrease detection limits, or a combination thereof. Whatever wavelengthis chosen to monitor emission, it can be referred to herein as theemission wavelength, or Emλ1.

In embodiments, an enzyme substrate can be chosen that has a Exλiso anda maximum emission (which can be utilized as Emλ1) that aresubstantially far apart in terms of wavelength (nanometers). Inembodiments, an enzyme substrate can be chosen that has a Exλiso and amaximum emission (as Emλ1) that are at least about 20 nm apart. Inembodiments, an enzyme substrate can be chosen that has a Exλiso and amaximum emission (as Emλ1) that are at least about 30 nm apart. Inembodiments, an enzyme substrate can be chosen that has a Exλiso and amaximum emission (as Emλ1) that are at least about 40 nm apart.

The particular wavelength at which emission is monitored (Emλ1) can alsobe chosen based in part on emission of the enzyme substrate. Forexample, in embodiments where the enzyme substrate absorbs at theisosbestic point but has an emission spectra that is different from thefluorescently detectable product, excitation at Exλiso along withdetection at Emλ1, where Emλ1 is chosen so that the enzyme substratedoes not emit (or does not substantially emit), can be advantageous.Such a scenario allows for pH independent detection of the fluorescentlydetectable product but not the enzyme substrate. Different fluorescentlydetectable products (and/or different enzyme substrates) can renderdifferent wavelengths more advantageous for use as Emλ1. In embodiments,4-methylumbelliferone β-D-galactopyranoside (4-MUG),7-hydroxycoumarin-3-carboxylic acid methyl ester-β-D-galactopyranoside(MHCgal), or 7-hydroxycoumarin-3-carboxylic acid ethylester-β-D-galactopyranoside (EHCgal) can be excited at Exλiso and thefluorescently detectable product can be detected (Emλ1) at a commonlyutilized wavelength, 455 nm without substantial detection of the enzymesubstrate.

Once the emitted light has been detected (at Emλ1), various additionalsteps can optionally be carried out. One optional step is that theemitted light can be quantified in order to estimate the amount ofmicroorganisms in the test sample. This can be accomplished by comparingthe integrated intensity of emitted light from the test sample with theintegrated intensity of emitted light (under the same conditions) fromone or more than one standard samples having known quantities ofmicroorganisms. A relative comparison can also be carried out by, forexample, comparing the fluorescent intensity at two different times (forexample before and after a sterilization procedure).

Another additional step that can optionally be carried out is to form animage of the detected light. In an embodiment, a CCD (or other detector)that has a number of individually addressable photosensitive detectorelements can enable the collection of fluorescent data from the sensoror sensor array on a pixel by pixel basis. This array can be used incombination with an illumination source and proper collection optics toobtain an image of, for example sites of growing microbial colonies onan inoculated two dimensional surface (for example a 3M™ Petrifilm™plate) that includes enzyme substrates as disclosed herein. Theresulting electronic image can be transmitted to the processor assembly,where image analysis software can be used to enhance the contrast of theimage and to count the number of fluorescent spots automatically (or auser can count the number of spots manually).

FIG. 2 depicts exemplary embodiments of methods disclosed herein.Embodiments of disclosed methods can include step 201, incubating a testsample with enzyme substrate, step 203, exciting a fluorescentlydetectable product within the incubated test sample with light having awavelength of Exλiso, and step 205, detecting the emitted light at Emλ1.

An optional step, step 207, may be added to such a method before step201. Step 207 includes processing a sample to form a test sample; asdiscussed above, such processing can include filtering, digesting,extracting, and the like.

Another optional step, step 209, may be added to such a method afterstep 205. Step 209 includes quantifying the microorganisms in the testsample; as discussed above, such quantification can include use ofsamples of known concentrations of microorganisms for example, or can bea relative comparison (i.e., obtaining a more or less than result).

Another optional step, step 211, may be added to such a method afterstep 205. Step 211 includes forming an image of the emitted light; asdiscussed above, formation of an image can include use of addressabledetectors and collection optics. In embodiments where step 211 iscarried out, the method can further include step 213, which can becarried out after step 211. Step 213 includes counting microorganismcolonies from the image; as discussed above, such colony counting can bedone automatically by a processor in communication with the imageforming electronics or can be done manually by a user.

It should also be noted that any combination of the above discussedsteps can be carried out in methods disclosed herein. Furthermore, anycombination of discussed steps can be repeated using different testsamples (or the same test sample).

Methods disclosed herein can also include steps of irradiating theincubated samples at wavelengths in addition to Exλiso. One such exampleincludes irradiating the incubated sample at a wavelength of Exλ2 for atime sufficient for the fluorescently detectable product to emit light.The wavelength referred to herein as Exλ2 can be the absorption maximumof the basic species of the fluorescently detectable product, theabsorption maximum of the acidic species of the fluorescently detectableproduct, or some other wavelength. For example, the fluorescentlydetectable product 4-MU, has an absorption maximum of the basic speciesat about 360 nm, and an absorption maximum of the acidic species atabout 320 nm; the fluorescently detectable product CyU has an absorptionmaximum of the basic species of about 405 nm, and an absorption maximumof the acidic species at about 355 nm.

Excitation at Exλiso and Exλ2 for times sufficient to cause thefluorescently labeled product to emit light can cause emissions of thesame wavelength (it should also be noted that emissions at differentwavelengths may also occur). In embodiments, excitation at Exλiso andExλ2 are not done at the same time, but are separated in time. Detectionof the independent emitted light (caused by excitation at Exλiso andExλ2) can also occur separated in time. Once the separate emissions(caused by excitation at Exλiso and Exλ2) are detected they can beutilized in various different ways. In embodiments, the two emissionscan be utilized in the same fashion, as a way of double checking theresults.

In embodiments, the two emissions can be utilized to determine adimensionless number that can be utilized to provide a response thatdoes not depend on measuring absolute fluorescent intensity. This can beaccomplished by determining a ratio that is the ratio of the emissioncaused by Exλiso and the emission caused by Exλ2. The numerator anddenominator choice is irrelevant; however if the choice remainsconstant, the ratio can be used to compare results from sample tosample.

In embodiments, another relative method can be utilized wherebyvariation in the excitation intensity (Exλiso) can be compensated for bymonitoring the excitation intensity while integrating the emissionsignal (Emλ1). Such an embodiment can be utilized along with the 3M™Attest™ Biological Monitoring System. In embodiments of such a methodutilized along with the 3M™ Attest™ Biological Monitoring System, thetwo groups of data can be mathematically processed in a microprocessorin the 3M™ Rapid Attest™ Autoreader™ (3M Co., St. Paul, Minn.).

In embodiments, exciting the incubated sample at two differentexcitation wavelengths (Exλiso and Exλ2) can be utilized to not onlydetermine the amount of microorganism in the test sample but also tomonitor the pH of the incubated sample over time. Such is the casebecause Exλ2 in essence monitors the amount of the acidic or basicspecies (assuming that a wavelength where at least one or the speciesabsorbs is utilized as Exλ2) as a function of time. Monitoring the pHcan be another method of monitoring microorganism growth, metabolicactivity, or a combination thereof.

FIG. 3 depicts exemplary embodiments of methods disclosed herein.Embodiments of disclosed methods can include step 301, incubating a testsample with enzyme substrate, step 303, exciting a fluorescentlydetectable product within the incubated test sample with light having awavelength of Exλiso, step 305, detecting the light emitted (Emλ1) as aresult of Exλiso, step 307, exciting a fluorescently detectable productwithin the incubated test sample with light having a wavelength of Exλ2,and step 309, detecting the light emitted (Emλ1) as a result of Exλ2. Inembodiments, step 307 and step 309 can be performed prior to steps 303and 305.

An optional step, step 311, may be added to such a method after step309. Step 311 includes determining a ratio of the emission caused byExλiso and the emission caused by Exλ2. Such a ratio can serve thepurpose of providing a dimensionless number that is not based onfluorescent intensity. Optional steps such as those discussed above withrespect to FIG. 2 may also be included in methods such as thoseexemplified in FIG. 3. It should also be noted that any combination ofthe above discussed steps can be carried out in methods disclosedherein. Furthermore, any combination of discussed steps can be repeatedusing different test samples (or the same test sample).

In embodiments, the ratio of the emission at Exλiso and Exλ2 canindicate things other than the amount of organisms in the sample. Inembodiments where the enzyme substrate emits strongly when excited atExλiso (for example, TUgal), the ratio of the emission at Exλiso andExλ2 can indicate the amount of organisms present because the ratio willbe largely invariant with enzymatic activity (unlike excitation near theanion max). In embodiments where there is minimal interference from theenzyme substrate at either excitation frequency (Exλiso and Exλ2) thenthe ratio of emission from Exλiso and Exλ2 can indicate a pH level. Forsome organisms that generate acid, a change in pH is indicative of theirpresence.

Methods disclosed herein can also include optional steps of detectingdifferent wavelengths of emission. As discussed above, once the sampleis irradiated with light having a wavelength of Exλiso, thefluorescently detectable product will emit light. As seen in FIG. 1C,for example, the fluorescently detectable product can emit light acrossa wide range of wavelengths. Methods disclosed herein include a step ofdetecting emitted light of a wavelength of Emλ1, such methods canoptionally include a further step of detecting emitted light at asecond, different wavelength. The additional wavelength of emitted lightthat can be detected is referred to herein as Emλ2.

Emλ2 can be any desired wavelength, and can be chosen based in part onthe intensity of the emissions at various wavelengths, the particulardetector (cost, etc.) that can be utilized, interference from othercomponents in the sample (for example the enzyme substrate), thewavelength of Emλ1 or a combination thereof. In embodiments, Emλ2 can bethe wavelength at which the fluorescently detectable product has itsmaximum intensity emissions (if such a wavelength were not utilized asEmλ1). Such an Emλ2 can be useful to minimize possible interference frombackground signals, decrease detection limits, or a combination thereof.In embodiments, Emλ2 can be the wavelength at which the emission of thefluorescently detectable product is independent of pH, or stated anotherway the isosbestic emission point (Emλiso). Such an Emλ2 can be usefulto compensate for a weak photoacid that has significant emission fromthe acidic and basic species, which would otherwise give a pH dependentresponse.

In embodiments, Emλ2 is chosen so that the amount of light emitted bythe fluorescently detectable product is at least greater than the amountof light emitted by the enzyme substrate. In embodiments, Emλ2 is chosenso that the amount of light emitted by the fluorescently detectableproduct is substantially greater than the amount of light emitted by theenzyme substrate. Such an Emλ2 can be useful to minimize the backgroundinterference from the enzyme substrate that has not been cleaved byenzymes within microorganisms.

In embodiments, detection at Emλ1 and Emλ2 are not done at the sametime, but are separated in time. Excitation of the fluorescentlydetectable product (by Exλiso for example) can also occur separated intime. The emissions at Emλ1 and Emλ2 can be utilized in variousdifferent ways. In embodiments, the two emissions can be utilized in thesame fashion, as a way of double checking the results.

In embodiments, the two emissions can be utilized to determine adimensionless number that can be utilized to provide a response thatdoes not depend on measuring absolute fluorescent intensity. This can beaccomplished by determining a ratio that is the ratio of the emission atEmλ1 and the emission at Emλ2. The numerator and denominator choice isirrelevant; however if the choice remains constant, the ratio can beused to compare results from sample to sample.

FIG. 4 depicts exemplary embodiments of methods disclosed herein.Embodiments of disclosed methods can include step 401, incubating a testsample with enzyme substrate, step 403, exciting a fluorescentlydetectable product within the incubated test sample with light having awavelength of Exλiso, step 405, detecting the light emitted at Emλ1 as aresult of Exλiso, step 407, exciting a fluorescently detectable productwithin the incubated test sample with light having a wavelength ofExλiso, and step 409, detecting the light emitted at Emλ2 as a result ofExλiso.

An optional step, step 411, may be added to such a method after step409. Step 411 includes determining a ratio of Emλ1 and Emλ2. Optionalsteps such as those discussed above with respect to FIG. 2 may also beincluded in methods such as those exemplified by FIG. 4. It should alsobe noted that any combination of the above discussed steps can becarried out in methods disclosed herein. Furthermore, any combination ofdiscussed steps can be repeated using different test samples (or thesame test sample).

Kits are also disclosed herein. Kits as disclosed herein can includeenzyme substrate, and a light source. The enzyme substrate as discussedabove can be included in disclosed kits. Generally, the enzyme substrateincludes an enzymatically hydrolysable group and a fluorescent group,wherein microorganisms present in the test sample include an enzyme thathydrolyzes the hydrolysable group from the fluorescent group to form afluorescently detectable product wherein the fluorescently detectableproduct has both an acidic and basic species. In embodiments, the enzymesubstrate can be more specifically defined as above. In embodiments, theenzyme substrate can be included as a component of growth media whichcan also be included in a disclosed kit.

Kits as disclosed herein also include a light source. The light sourceis at least capable of providing light having a wavelength of Exλiso.Exemplary light sources can include a visible laser diode, a visiblelight emitting diode (LED), an incandescent filament, an organic lightemitting diode (OLED), or any other suitable light source. Inembodiments, inexpensive LEDs can be utilized as a light source withindisclosed kits. The light source can also be combined with or used incombination with various filters and optics as are commonly utilized.

The light source included in the kit can also be capable of providinglight having a wavelength of Exλ2, where Exλ2 can be the absorptionmaximum of the basic species of the fluorescently detectable product,the absorption maximum of the acidic species of the fluorescentlydetectable product, or some other wavelength. Alternatively a secondlight source capable of providing light having a wavelength of Exλ2 canbe included in disclosed kits. In the latter embodiments, the kit caninclude at least two light sources, one capable of providing lighthaving a wavelength of Exλiso; and a second capable of providing lighthaving a wavelength of Exλ2, where Exλ2 can be the absorption maximum ofthe basic species of the fluorescently detectable product, theabsorption maximum of the acidic species of the fluorescently detectableproduct, or some other wavelength.

Kits as disclosed herein can also include containers configured to mixat least the enzyme substrate and the sample to be tested. Inembodiments, the optional container can come preloaded with the enzymesubstrate; or in embodiments, the optional container can come preloadedwith a growth media containing the enzyme substrate. An example of acontainer that can be preloaded with a growth media containing theenzyme substrate is 3M™ Petrifilm™ plates (3M Co., St. Paul, Minn.).Another example of a container that can be preloaded with a growth mediacontaining enzyme substrate is 3M™ Attest™ Biological Monitoring System(3M Co., St. Paul, Minn.). Alternatively, a container can optionally beprovided along with separately packaged enzyme substrate, whether mixedwith growth media or not.

Disclosed kits can also optionally include a detector. Detectors, suchas photomultiplier tubes, avalanche photodiodes, charge coupled devices(CCDs), photodiodes, or other active devices for example, may beutilized. The detector can also be combined with or used in combinationwith various filters and optics as are commonly utilized. A detectorthat can be optionally included in disclosed kits can be capable ofdetecting one or more than one wavelength of emitted light.Alternatively, more than one detector can optionally be included.

Disclosed kits can also optionally include other components includingfor example imaging components (for example for imaging the detectedemitted light), processor(s), or sample collection or preparation aids.In embodiments, one or more processors can be utilized or configured toprocess images from imaging components to clean up the images orautomatically count colonies; to provide output indicating the presenceor absence of microorganisms in the test sample; to control a lightsource(s), detector(s), optional components, or some combinationthereof; or some combination thereof.

Disclosed kits can be configured to work in concert with or can beincluded along with currently utilized systems such as the 3M™Petrifilm™ product line, the 3M™ Attest™ Biological Monitoring System,the SPECTRA MAX M5 (Molecular Devices, Sunnyvale, Calif.), and the 3M™Clean-Trace™ System (3M Co., St. Paul, Minn.).

In embodiments of disclosed kits, the fluorescently detectable productcan be a coumarin derivative. In embodiments, the fluorescentlydetectable product can be 4-methylumbelliferone and Exλiso (thewavelength the light source is capable of generating) can be about 330nm. In embodiments, the fluorescently detectable product can be3-cyano-7-hydroxycoumarin and Exλiso (the wavelength the light source iscapable of generating) can be about 375 nm. In embodiments, thefluorescently detectable product can be 7-hydroxycoumarin-3-carboxylicacid ester and Exλiso (the wavelength the light source is capable ofgenerating) can be about 370 nm. In embodiments, the fluorescentlydetectable product can be 3-(2-thienyl)umbelliferone and Exλiso (thewavelength the light source is capable of generating) can be about 380nm.

EXAMPLES

Materials and Methods

Unless otherwise noted, all chemicals were obtained from Aldrich andwere used without further purification.

All parts, percentages, ratios, etc. in the examples are by weight,unless noted otherwise. Solvents and other reagents used were obtainedfrom Sigma-Aldrich Chemical Company; Milwaukee, Wis. unless indicatedunless specified differently.

Materials

7-hydroxycoumarin-3-carboxylic acid ethyl ester (EHC) was preparedaccording to Chilvers et. al. J. Appl. Microbiology 2001, 91, 1118-1130.

3-(2-thienyl)umbelliferone (TU) was prepared as in U.S. Pat. No.6,372,895 (Bentsen et al.).

3-(2-thienyl)umbelliferone galactoside (TUgal) was prepared as in U.S.Pat. No. 6,372,895 (Bentsen et al.).

β-D-galactosidase was acquired from EMD Biosciences, Inc. (San Diego,Calif.).

Example 1

4-methylumbelliferone (4-MU) was dissolved in dimethyl sulfoxide (DMSO)at a concentration of 1 mg/mL. A 96-well plate was prepared with eachwell containing 100 μL of 100 mmolar phosphate buffer at pH valuesranging from 8.0 down to 2.5. 10 μL of the 4-MU solution was added toeach of the wells. A set of 10× diluted wells were also prepared toeliminate artifacts due to self-quenching of the fluorophore at highconcentrations. The 10× diluted wells were prepared by diluting the 1mg/mL solution to 0.1 mg/mL with DMSO and then adding 10 μL of thissolution to 100 μL of 100 mmolar phosphate buffer in each of the wells.

Absorption spectra from 250 to 500 nm were recorded using a SPECTRAMAXM5 (available from Molecular Devices, Sunnyvale, Calif.) fluorescenceplate reader. The results for the absorption spectra are shown FIG. 1A.These results demonstrated that the absorption of the neutral 4-MUspecies (formed at a low pH) is low at wavelengths near the absorptionmaxima for the anion (formed at a high pH). The absorption maximum ofthe neutral 4-MU occurs at about 320 nm and there is an isosbestic point(where absorbance is invariant with composition) at about 330 nm.

Emission at various wavelengths was also measured with the SPECTRAMAX M5fluorescence plate reader. FIG. 1C shows the resulting emission for the10× diluted samples as a function of emission wavelength for anexcitation wavelength of 331 nm and for various pH values. These resultsdemonstrate that excitation near the isosbestic point (about 330 nm)provides a fluorescent response that is largely invariant with pH valueover the range studied (2.5 to 8). The maximum emission occurred at awavelength of 450 nm.

For comparison, emission was also measured as a function of pH value foran excitation wavelength of 360 nm, which is near the maximum absorptionwavelength for the anion. FIG. 1B shows emission at about 450 nm as afunction of pH for an excitation wavelength of 360 nm and 331 nm(approximately at the isosbestic point).

Example 2

96-well plates were prepared as in Example 1, but with3-cyano-7-hydroxycoumarin (CyU) substituted for 4-MU. Absorption andemission spectra were measured as in Example 1.

Absorption spectra of CyU at various pH values were obtained (notshown). Emission spectra were determined at various pH values for CyU atthree different excitation wavelengths, corresponding to the maximumabsorbance of the neutral species (355 nm), the isosbestic point (375nm) (shown in FIG. 5A) or the anion absorbance maximum (405 nm). Thewavelength of maximum emission was about 455 nm when the excitationwavelength was 375 nm. FIG. 5B shows emission at a wavelength of 455 nmas a function of pH value for excitation wavelengths of 355 nm, 375 nm(approximately at the isosbestic point), and 405 nm. The fluorescencewas approximately independent of pH value when the excitation was at theisosbestic point.

The ratio of the peak emission intensity recorded at differentexcitation wavelengths was determined. As shown in FIG. 5C, this yieldsa dimensionless number that is sensitively dependent upon pH. Thisprovides a sensitive pH response that does not depend on measuringabsolute fluorescent intensity.

Example 3

7-hydroxycoumarin-3-carboxylic acid ethyl ester (EHC) was dissolved indimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL. A 96-well platewas prepared with each well containing 100 μL of 100 mmolar phosphatebuffer at pH values ranging from 8.0 down to 2.5. 10 μL of the EHCsolution was added to each of the wells. 10× diluted wells were preparedby taking 10 μL of the wells prepared at the initial concentration, andadding this to 90 μL of buffer at the appropriate concentration.Absorption and emission spectra were measured as in Example 1.

Absorption spectra of EHC at various pH values were obtained (notshown). The isosbestic point was found to occur at about 370 nm. Thefluorescence as a function of pH for the 10× diluted EHC samples areshown in FIG. 6 with an excitation wavelength of 370 nm and, forcomparison, with an excitation wavelength of 400 nm (anion maximumabsorbance). The fluorescence was approximately constant when excited atthe isosbestic point.

Example 4

96-well plates were prepared as in Example 1, but with3-(2-thienyl)umbelliferone (TU) substituted for 4-MU. TU is described inU.S. Pat. Nos. 6,372,895 (Bensten et al.) and 6,566,508 (Bensten et al.)which are hereby incorporated herein by reference. Absorption andemission spectra were measured as in Example 1, 2, and 3.

Absorption spectra of the 10×TU samples at various pH values are shownin FIG. 7A. The isosbestic point was found to occur at about 380 nm. Theabsorption peaks were generally 5-10 nm red-shifted compared to CyU andEHC.

Emission spectra were obtained for various pH values for TU at threedifferent excitation wavelengths, 405 nm (anion maximum, forcomparison), 380 nm (isosbestic point—shown in FIG. 7B), and 365 nm(neutral species maximum, for comparison). The anion emission band wasred-shifted 40 nm to about 495 nm compared to 455 nm for Examples 1 and2. The Stokes shift for the anionic species was determined to beapproximately 90 nm.

When samples at low pH were excited at 405 nm (anion maximum absorbance)the emission at 495 nm diminished and the peak maximum was blue-shiftedabout 30 nm to 465 nm. The blue shift was more apparent when theexcitation wavelength was set to the isosbestic point (380 nm, FIG. 7B)or the neutral species maximum (365 nm). These results indicate that atlow pH values, there is pronounced emission from the excited state ofthe neutral TU, in addition to that from the photogenerated anion.

Although there was variation in the emission spectra with varying pHvalues when TU was excited at the isosbestic point for absorption, therewas an isosbestic point for emission (about 490 nm, see FIG. 7B) wherethe emission did not depend on the pH. The fluorescence as a function ofpH for the TU samples are shown in FIG. 7C for excitation wavelengths of380 nm and 405 nm and for emission wavelengths of 490 nm and 500 nm. Thefluorescence was approximately independent of pH when excited at theabsorption isosbestic point (380 nm) and measured at the emissionisosbestic point (490 nm).

Example 5

4-methylumbelliferone phosphate disodium salt (4-MUP) was dissolved inwater at a concentration of 1 mg/mL. A 96-well plate was prepared witheach well containing 100 μL of 100 mmolar phosphate buffer at pH valuesranging from 8.0 down to 2.5. 10 μL of the MUP solution was added toeach of the wells. A set of 10× diluted wells were also prepared toeliminate artifacts due to self-quenching of the fluorophore at highconcentrations. The 10× diluted wells were prepared by diluting the 1mg/mL solution to 0.1 mg/mL with water and then adding 10 μL of thissolution to 100 μL of 100 mmolar phosphate buffer in each of the wells.Absorption and emission spectra were measured as in Example 1.

It was found that 4-MUP had noticeable absorption at the isosbesticpoint of the 4-MU dye (about 330 nm), but minimal absorption at theanion maximum (about 360 nm). The MUP absorption spectrum showed littlepH-dependence and the absorption at the isosbestic point was only about¼ to % of that of the free dye.

The maximum emission occurred at about 390 nm for the range of pH valuesconsidered. The emission was very small at the characteristic emissionwavelength of 4-MU (455 nm). This means that the presence of unreacted4-MUP would not significantly increase the background signal when thepresence of 4-MU is measured.

Example 6

4-methylumbelliferone-β-D-galactoside (4-MUG) was dissolved in dimethylsulfoxide (DMSO) at a concentration of 0.1 mg/mL. A 96-well plate wasprepared with each well containing 100 μL of 100 mmolar phosphate bufferat pH values ranging from 2.5 (column 1) up to 8.0 (column 12). 10 μL ofthe 4-MUG solution was added to each well in the first four rows. Two ofthese rows were further treated with 10 μL of a reagent solutioncontaining mercaptoethanol (1.4% v/v), MgCl₂ (2% w/w) and(3-D-galactosidase (80 μg/mL). 10 μL of a 0.1 mg/mL solution of 4-MU inDMSO was added to two more rows for comparison. Absorption and emissionspectra were measured as in Example 1.

The absorption spectra of 4-MUG with and without galactosidase wereobtained. The spectrum of 4-MUG in the absence of added(3-D-galactosidase showed little pH-dependence and was similar to thatof 4-MU in acid solution, i.e.: that of the neutral 4-MU. Consequently4-MUG absorbs strongly at the isosbestic point of 4-MU dye (about 330nm), but minimally at the anion maximum (about 360 nm).

The emission spectra (335 nm excitation) for 4-MUG in the absence andpresence of β-D-galactosidase were also obtained for pH values from 2.5to 8. The maximum emission was low and occurred at about 390 nm in theabsence of enzyme, with very little intensity at the characteristicemission wavelength of 4-MU (455 nm). In the presence of theβ-D-galactosidase enzyme, an emission peak was observed at 455 nmcorresponding to emission from the free 4-MU anion.

The emission at 455 nm (with an excitation at the isosbestic wavelengthof 355 nm) for 4-MU, as well as 4-MUG in the absence and presence ofβ-D-galactosidase is shown in FIG. 8A as a function of pH. The emissionfor 4-MUG+β-D-galactosidase falls off slowly below pH 7, consistent withthe reported activity of the enzyme (Sungur and Akbulut, J. Chem. TechBiotechnol. 1994, 59, 303-306). Nevertheless, useful intensity wasobserved at least as low as pH 5. For comparison, emission resultingfrom excitation at the anion maximum (about 360 nm) is shown in FIG. 8B.In this case, emission was very low at acidic pH values and increasedrapidly above pH 7.

This example demonstrated the ability to use MUG to effectively detectβ-D-galactosidase over a wide range of pH values without adjustingexcitation or emission frequencies by exciting at the isosbestic pointof the dye.

Example 7

Methyl 7-hydroxycoumarin-3-carboxylate galactoside (MHCgal) was preparedby reacting EHC with α-acetobromo-D-galactose and then hydrolyzing theprotected galactoside with sodium methoxide, as described in Chilvers(J. Appl. Microbiology 2001, 91, 1118-1130). MHCgal was dissolved indimethyl sulfoxide (DMSO) at a concentration of 1.0 mg/mL, and then 10μL put in each well of a 96-well plate prepared with wells containing100 μL of 100 mmolar phosphate buffer at pH levels ranging from 8.0 downto 2.5. Half of the rows were further treated with 10 μL of a reagentsolution containing mercaptoethanol (1.4% v/v), MgCl₂ (2% w/w) andβ-D-galactosidase (80 μg/mL). 10 μL from each well of this plate wasthen added to the corresponding well of another plate containing 100 μLof the phosphate buffer with the appropriate pH in the wells creating a10× dilution plate. Absorption and emission spectra were measured as inExample 1.

The absorption spectra of MHCgal with and without β-D-galactosidase wereobtained. The spectrum of MHCgal in the absence of addedβ-D-galactosidase showed no pH-dependence over the pH range tested, anddemonstrated a maximum absorbance at about 340 nm. With addedβ-D-galactosidase, the absorption shifted to 350 nm for pH 5-6 and to400 nm for alkaline values. These are consistent with the absorptionmaxima for the neutral and anionic EHC species.

The emission spectra for MHCgal (with an excitation wavelength of 370nm) in the absence and presence of β-D-galactosidase were obtained forpH values from 2.5 to 8. The emission was low without enzyme, with verylittle emission at the characteristic emission wavelength of simplecomuarins (˜450 nm). In the presence of the β-D-galactosidase enzyme, astrong emission peak was observed at 445 nm corresponding to emissionfrom free MHC anion.

The emission at 445 nm (for excitation at the 370 nm isosbestic point,and, for comparison, for excitation at the anion maximum at 400 nm) forMHCgal in the absence and presence of β-D-galactosidase is shown in FIG.9 as a function of pH. In the presence of β-D-galactosidase, theemission of MHCgal when excited at 370 nm is substantial and constantfrom a pH of about 5 and higher. In contrast, the excitation at theanion maximum resulted in emission that was very low at low pH valuesbut increased steeply from pH 6.

This example demonstrated the ability to use MHCgal to effectivelydetect β-D-galactosidase over a wide range of pH values withoutadjusting excitation or emission frequencies by exciting at theisosbestic point of the dye.

Comparative Example 8

3-(2-thienyl)umbelliferone galactoside (TUgal) was dissolved in dimethylsulfoxide (DMSO) at a concentration of 0.1 mg/mL. A 96-well plate wasprepared with wells containing 100 μL of 100 mmolar phosphate buffer atpH levels ranging from 8.0 down to 2.5. 10 μL of the TUgal solution wasadded to each of the wells, and then half of the rows were furthertreated with 10 μL of a reagent solution containing mercaptoethanol(1.4% v/v), MgCl₂ (2% w/w) and β-galactosidase (80 μg/mL). Absorptionand emission spectra were measured as in Example 1.

The emission for TUgal at 490 nm (380 nm excitation) and 500 nm (410 nmexcitation) in the presence and absence of β-D-galactosidase is shown inFIG. 10 as a function of pH. When measured at the isosbestic frequenciesfor TU (380 ex, 490 em; see Example 4) TUgal possessed comparableemission to TU, the product fluorophore itself. Thus there was littledifference between emission in the presence or absence of enzyme. Themeasurement of fluorescence near the anion maxima (ex 410 nm, em 500 nm)reduced the emission of the enzyme substrate well below that of theanion and enabled the use of TUgal as a β-D-glactosidase substrate.

As seen in this example, irradiation at the isosbestic excitationfrequency can be less effective in situations where it causes the enzymesubstrate to emit strongly at the same frequencies as the freefluorophore.

Thus, embodiments of methods of detecting microorganisms and kitstherefore are disclosed. One skilled in the art will appreciate that thepresent disclosure can be practiced with embodiments other than thosedisclosed. The disclosed embodiments are presented for purposes ofillustration and not limitation, and the present disclosure is limitedonly by the claims that follow.

What is claimed is:
 1. A method of determining the amount ofmicroorganisms present in a test sample, the method comprising the stepsof: a) incubating the test sample with enzyme substrate to form anincubated sample, wherein the enzyme substrate comprises anenzymatically hydrolysable group and a fluorescent group, whereinmicroorganisms present in the test sample include an enzyme thathydrolyzes the hydrolysable group from the fluorescent group to form afluorescently detectable product, and wherein the fluorescentlydetectable product has both an acidic and basic species; b) exciting thefluorescently detectable product with light having a wavelength ofExλiso for a time sufficient for the fluorescently detectable product toemit light, wherein Exλiso is the absorbance isosbestic point of thefluorescently detectable product; c) detecting light emitted at awavelength of Emλ1; and d) quantifying the light emitted at thewavelength of Emλ1, wherein the quantity of the light emitted at thewavelength Emλ1 is indicative of the amount of microorganisms present inthe incubated test sample, wherein the quantity of the light emitted atthe wavelength Emλ1 is substantially constant across a pH range of 2.5to 8.0.
 2. The method according to claim 1, wherein the pH of theincubated sample is not adjusted before the excitation and detectionsteps.
 3. The method according to claim 1 further comprising excitingthe fluorescently detectable product with light having a wavelength ofExλ2 for a time sufficient for the fluorescently detectable product toemit light, wherein Exλ2 is the absorption maximum of the basic speciesof the fluorescently detectable product or the absorption maximum of theacidic species of the fluorescently detectable product.
 4. The methodaccording to claim 3 further comprising determining the ratio of theemission caused by excitation at Exλiso and the emission caused byexcitation at Exλ2, wherein the ratio is indicative of the amount ofmicroorganisms present in the test sample.
 5. The method according toclaim 1, wherein the fluorescently detectable product is a coumarinderivative.
 6. The method according to claim 1, wherein thefluorescently detectable product is 4-methylumbelliferone and Exλiso isabout 330 nm.
 7. The method according to claim 1, wherein thefluorescently detectable product is 3-cyano-7-hydroxycoumarin and Exλisois about 375 nm; the fluorescently detectable product is7-hydroxycoumarin-3-carboxylic acid ethyl ester and Exλiso is about 370nm; or the fluorescently detectable product is7-hydroxycoumarin-3-carboxylic acid methyl ester and Exλiso is about 370nm.
 8. The method according to claim 1, wherein the fluorescentlydetectable product is 3-(2-thienyl)umbelliferone and Exλiso is about 380nm.
 9. The method according to claim 1, wherein the step of incubatingthe test sample with the enzyme substrate comprises adding the sample ina liquid form to a dehydrated growth media containing the enzymesubstrate.
 10. The method according to claim 1 further comprisingforming an image of the detected light.
 11. The method according toclaim 10 further comprising counting colonies from the image.
 12. Themethod according to claim 1, wherein Emλ1 is the wavelength at themaximum fluorescence of the fluorescently detectable product.
 13. Themethod according to claim 1 further comprising quantifying the lightemitted near a wavelength Emλ2, wherein Emλ1 and Emλ2 are differentwavelengths, and further comprising determining a ratio of the lightemitted at a wavelength Emλ1 and the light emitted at a wavelength Emλ2,wherein the ratio is indicative of the amount of microorganisms presentin the test sample.
 14. The method of claim 13, wherein the wavelengthEmλ2 is the wavelength of the isosbestic emission point.
 15. The methodof claim 13, wherein the amount of light emitted by the fluorescentlydetectable product near the wavelength Emλ2 is substantially greaterthan the amount of any light emitted by the enzyme substrate near thewavelength Emλ2.
 16. A method of determining the amount ofmicroorganisms present in a test sample, the method comprising the stepsof: a) incubating the test sample with an enzyme substrate to form anincubated sample, wherein the enzyme substrate comprises anenzymatically hydrolysable group and a fluorescent group, whereinmicroorganisms present in the test sample include an enzyme thathydrolyzes the hydrolysable group from the fluorescent group to form afluorescently detectable product, and wherein the fluorescentlydetectable product has both an acidic and basic species; b) exciting thefluorescently detectable product with light having a wavelength ofExλiso for a time sufficient for the fluorescently detectable product toemit light, wherein Exλiso is the isosbestic point of the fluorescentlydetectable product; c) detecting light emitted at a wavelength of Emλ1as a result of the excitation with light having a wavelength of Exλiso;d) exciting the fluorescently detectable product with light having awavelength of Exλ2 for a time sufficient for the fluorescentlydetectable product to emit light, wherein Exλ2 is the absorption maximumof the basic species of the fluorescently detectable product or theabsorption maximum of the acidic species of the fluorescently detectableproduct; e) detecting light emitted at a wavelength of Emλ1 as a resultof the excitation with light having a wavelength of Exλ2; and f)calculating a ratio based on the light emitted at a wavelength of Emλ1as a result of the Exλiso excitation light and the light emitted as aresult of the Exλ2 excitation light, wherein the ratio is indicative ofthe amount of microorganisms present in the test sample.
 17. The methodaccording to claim 16, wherein Exλ2 is the absorption maximum of theacidic species of the fluorescently detectable product.
 18. The methodaccording to claim 16, wherein the ratio can be utilized to monitor thepH of the incubated sample over time.